PHYSIOLOGICALLY INDUCED CHANGES IN ADRENOCORTICAL MITOCHONDRIA

1. Adrenocortical mitochondria contain filamentous or saccular internal membranes and are probably the sites of lipide elaboration or accumulation. They are numerous in the stimulated gland and scanty after hypophysectomy. There is evidence that they are expendable: light cells contain numbers of irregularly vacuolated and broken mitochondria with disorganized internal membranes. 2. Openings have been observed in some mitochondrial limiting membranes, particularly in the stimulated hamster and rat adrenal cortices, but to a lesser extent in the unstimulated glands. 3. Similarities between mitochondrial and Golgi membranes suggest a possible mode of mitochondrial formation. 4. These findings have been interrelated and discussed.     

Morphology and ultrastructure of the convoluted gland in the ant Dinoponera australis (Hymenoptera: Formicidae)

The morphology and fine structure of the convoluted gland inside the venom reservoir of the ponerine ant Dinoponera australis (Hymenoptera: Formicidae) are described. The cells of the convoluted gland can be divided into 3 major groups: (1) epithelial cells, (2) glandular cells with end apparatus secreting into the tubule inside the convoluted gland, and (3) glandular cells with end apparatus secreting directly into the venom reservoir. A fourth group of cells belonging to the venom gland of this ant is also discussed as (4) secretory cells of the free tubule (not a part of the convoluted gland). The epithelial cells in the convoluted gland do not have many organelles. Most cells of group 3 are characterized by numerous mitochondria. In some of these cells, the mitochondria possess tubular cristae. Tubule cells of group 2 inside the convoluted gland, possess little rough endoplasmic reticulum when compared with cells of group 4, situated in the free tubule.     

Further studies on corticosteroidogenesis VI. Pyruvate and malate supported steroid 11β-hydroxylation in rat adrenal gland mitochondria

Pyruvate and pyruvate plus ATP have been shown to support 11β-hydroxylation of 11-deoxycorticosterone into corticosterone in incubated rat adrenal gland mitochondria. Corticosterone production with pyruvate plus ATP was not as great as with malate plus P_i, malate plus ATP or malate plus pyruvate. Respiratory chain inhibitors, trans-aconitate, oxaloacetate, arsenite and the uncoupler 2,4-dinitrophenol, inhibited corticosterone formation. On the other hand, cysteine sulfinate and pyruvate, which led to the removal of excess metabolic oxaloacetate formed from malate oxidation, increased rat adrenal mitochondrial O₂ consumption as well as corticosterone production from 11-deoxycorticosterone. P_i and ATP also appeared to act in the same way in that these agents brought about a greater conversion rate of oxaloacetate into pyruvate. Pyruvate, resulting from the oxidation of malate, accumulated in the incubation system only when arsenite was added. Arsenite additions to malate and isocitrate inhibited the conversion of 11-deoxycorticosterone into corticosterone except when the 11β-hydroxylation of 11-deoxycorticosterone was supported with exogenous NADPH in Ca²⁺-swollen mitochondria. These results as well as the observations that NAD-linked malate dehydrogenase ( -malate: NAD⁺ oxidoreductase (decarboxylating), EC 1.1.1.39) is at least 10 times as active as the NADP-linked enzyme ( -malate: NADP⁺ oxidoreductase (decarboxylating), EC 1.1.1.39) in sonicated rat adrenal gland mitochondria, led to the conclusion that under our incubation conditions malate was mainly oxidized via the NAD-linked malate dehydrogenase. The fact that in malate incubations pyruvate did not accumulate because of its further metabolism in rat adrenal gland mitochondria, does not support the possibility that these mitochondria are the source of pyruvate for a “malate shuttle” originally thought to occur in rat adrenal gland⁷. This shuttle would have depended on the formation of pyruvate from malate in rat adrenal gland mitochondria followed by extrusion of the pyruvate formed intramitochondrially into the cytoplasm of the cell.     

Structural studies of the adrenal zona glomerulosa and renal juxtaglomerular apparatus in pregnant sheep

The ultrastructural changes in the adrenal zona glomerulosa and renal juxtaglomerular apparatus have been examined during normal pregnancy in sheep. As pregnancy progressed, increasing numbers of cells in the adrenal zona glomerulosa displayed mitochondria with straight tubular "rod-like" structures replacing their normal lamelliform cristae; groups of cells showing these mitochondrial changes were predominantly located in the middle and superficial regions of the zona glomerulosa, but at all stages remained interspersed with cells with apparently normal mitochondria. In the same animals, the renal juxtaglomerular index was raised, reflecting an increase in renin storage, and juxtaglomerular myoepithelioid cells showed increased numbers of cytoplasmic granules, but no apparent increase in granular endoplasmic reticulum and Golgi profiles; there were no distinguishing morphological changes in juxtaglomerular peripolar cells. These findings provide morphologic evidence of stimulation of the adrenal zona glomerulosa in association with increased juxtaglomerular renin storage during pregnancy. The mitochondrial changes observed in an increasing proportion of cells in the zona glomerulosa closely resemble those seen in sodium-depleted animals, and may reflect the altered steroidogenic capacity of the adrenal gland in pregnant sheep. The finding of groups of cells displaying altered mitochondria lying next to cells with normal mitochondria suggests the presence of cells with different sensitivities to stimuli for aldosterone production or may indicate the presence of different cell types in the zona glomerulosa responding to different stimuli.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. I: Topology of bovine adrenal cortex mitochondria in the orthodox configuration

Bovine adrenal cortex mitochondria examined by electron microscopyin situ orin vitro in 0·25 M sucrose have an unusual cristal membrane structure. The cristae usually appear as unconnected vesicles within a double membrane system. A few of the vesicles appear to be attached to the inner boundary membrane or to one or more other vesicles. The configuration of such mitochondria will be defined as the orthodox configuration. In this communication we will provide evidence that the inner membrane is not composed of multiple vesicles, but is one continuous membrane with tubular invaginations, and that these invaginations alternately are ballooned out and squeezed down. A mechanism has been proposed to account for the differentiated structure of the cristae of adrenal cortex mitochondria.     

Structural characteristics and innervation of chromaffin tissue in the adrenal gland of the axolotl

Each adrenal gland of the Axolotl consists of a strip lying all along the medio-lateral edge on the ventral surface of the kidney. The gland is composed of interrenal cells (IC) and chromaffin cells (CC). The IC contained a great number of pleomorphic lipid droplets, smooth endoplasmic reticulum and elongated mitochondria with tubulo-vesicular cristae. Two types of CC, always disposed in clusters and exhibiting long cytoplasmic processes were described according to the electron density, size and shape of granules distributed in their cytoplasm; noradrenaline cells (NA) and adrenaline cells (A). The innervation and ultrastructural differences from the adrenal gland of other Anurans were discussed.     

Ultrastructure of rat ovarian interstitial gland cells during pregnancy

Summary The fine structure of the interstitial gland of the rat ovary was studied at estrus and on Days 4, 6, 10, 14 and 18 of pregnancy. At estrus, ovarian interstitial cells have small nuclei with dense irregular clumps of heterochromatin. Mitochondria are small and rod-shaped and have predominantely lamellar cristae. Numerous osmiophilic lipid droplets are present. At Days 4 and 6, nuclear heterochromatin is reduced, and nucleoli are larger and complex. Mitochondria are enlarged and often bizarre-shaped and have tubular cristae. Golgi and smooth endoplasmic reticulum are more conspicuous. At Day 10, prominent ultrastructural features include nuclei with conspicuous heterochromatin, smaller mitochondria with both lamellar and tubular cristae, numerous ribosomes and lipid droplets with decreased osmiophilia. At Days 14 and 18, nuclei have increased heterochromatin, mitochondria are small and have lamellated cristae and an increase in the size and number of lipid droplets occurs. These observations suggest that steroidogenic activity of interstitial cells is highest during the first half of pregnancy and regresses during the last half. It is suggested that the interstitial gland is an important ovarian source of pregnancy hormone(s) during the first half of gestation and that LH may modulate steroidogenic activity in this ovarian component.     

Ultrastructure of the cortical cell of the interrenal gland of the American bullfrog (Rana catesbeiana)

Summary The cortical cell of the interrenal gland of the American bullfrog, Rana catesbeiana, was examined in the electron microscope. These cells occur in small groups and cords and are quite irregular in shape. The cortical cell is reminiscent of adrenal cortical cells from other vertebrates. Liposomes are variable in size and density. Smooth endoplasmic reticulum is scant in amount and predominantly of the fine tubular type. Mitochondria have vesicular cristae, a dense matrix, and occasionally have blebs, vacuoles, and myelin-like whorls at their surfaces. Intimate morphological relationships are found among the Golgi apparatus, lysosomes, and liposomes, and among Golgi vacuoles, mitochondria, and liposomes. In addition microfibrils are a prominent feature of the cortical cell. The biochemical events of steroidogenesis in amphibia and other vertebrates are discussed in relationship to the organellar interrelations found in the bullfrog interrenal cortical cells. Based on the available chemical and morphological information a scheme is proposed of movement of the steroidal intermediates through the cell that tentatively identifies the localizations of the various enzyme systems involved in corticosteroidogenesis from acetate to corticosterone and aldosterone.Supported in part by N.I.H. Grant RR 06138. Health Sciences Advancement Award.     

ULTRASTRUCTURAL ZONATION OF ADRENOCORTEX IN THE RAT

The fine structure of the different zones in the adrenal cortex of the adult rat has been studied under the electron microscope. Four regions mainly differentiated by the mitochondrial morphology, the lipid droplets, and the structure of the ground cytoplasm were recognized. In the glomerular zone mitochondria are thin and elongated with an abundant matrix. The inner structure is characterized by the presence of tubules of 300 A that are straight or bend at an angle and which may be grouped in parallel array giving a pseudocrystalline pattern. The wall of each tubule is a finger-like projection of the inner membrane and its cavity corresponds to the outer chamber of the mitochondrion. In the intermediary zone mitochondria are larger and irregular. The matrix is filled with convoluted tubules and vesicular elements. The lipid droplets are larger and irregular in the glomerulosa and and small in the intermedia. The ground substance is dense and contains free ribosomes in the glomerulosa and starts to be vacuolated in the intermedia. In the fasciculata mitochondria are round or oval and are filled with vesicular elements with a mean size of 450 A. Larger vesicles and more clear elements (vacuoles) are seen near the edge as if their content was diluted. Some of these vacuoles protrude on the surface. In the reticular zone mitochondria are also vesicular but frequently show signs of alteration and disruption. Dense elements recognized as microbodies are observed in the fasciculata but they increase in number in the reticularis. These results are discussed on the light of the so called zonal theory of the adrenal cortex. Two stages in the differentiation of the mitochondria are postulated. The tubular structure of the glomerulosa undergoes a process of disorientation and dilatation of the tubules to form the tubulo-vesicular elements of the intermediary zone. In a second stage of differentiation, by fragmentation of the tubules, the vesicular structure of fasciculata is formed. These findings are discussed from the viewpoint of the relationship between mitochondria and synthesis of steroid hormones. A secretory process that starts within mitochondria by the formation of vesicles and proceeds into the ground cytoplasm, as extruded and more clear vacuoles, is postulated.     

Possible relationship between the activity of the adrenal gland and the subcommissural organ in the lizard Lacerta s. sicula raf.

Summary In order to study the possible functional relationship between the adrenal gland and the subcommissural organ (SCO) in the lizard Lacerta s. sicula Raf., ACTH was administered to some specimens of this species in January when both the adrenal gland and the subcommissural organ have a very low activity. In comparison to untreated controls, the adrenals of animals treated with ACTH showed clear signs of stimulation, presenting enlarged blood vessels, very few lipid droplets, numerous polymorphic mitochondria and abundant tubular smooth endoplasmic reticulum. In addition, a distinct increase in secretory material was observed in the subcommissural cells of specimens treated with ACTH. These cells showed large cisternae of the rough endoplasmic reticulum filled with granular material in the basal region, numerous secretory granules of two types in the apical region and a reduced number of microvilli on the free cell surface. These findings, together with the results of preceding studies, lead the authors to the consideration that steroid hormones might play a role in the regulation of the secretory activity of the SCO.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Clasper gland morphology and development in Potamotrygon magdalenae (Elasmobranchii: Potamotrygonidae)

Clasper gland morphology and development in Potamotrygon magdalenae and its relation with the acquisition of reproductive maturity is described in males of different developmental stages (embryos, neonates, juveniles, and reproductively active and resting adults). The glands are subcutaneous masses in the proximal base of each clasper. They are partially bilobate organs with a ventral groove that bears a row of papillae. Glands tend to be asymmetric, the left gland has a larger size, a trend that has been observed in other organs of elasmobranchs. Glands are formed by radially organized tubular secretory units lined with a simple columnar epithelium with basal nuclei and granular eosinophilic cytoplasm; vascularized loose connective tissue surrounds the gland units. The gland is covered by two layers of striated muscle tissue in circular and longitudinal arrangement. The clasper glands begin to develop in neonates and their secretory activity begins in juveniles. The active secretion of the clasper gland is observed in mature males, it includes glycoproteins and sulfated mucopolysaccharides. The size of the glands has a positive and direct relationship with body size, measured as disc width. Significant differences in clasper gland size were found between mature (active and resting) and immature (neonates and juveniles) males, suggesting that the acquisition of the sexual maturity involves the increase in the size of the gland due to a highly augmented secretory activity. Therefore, clasper glands are clearly associated with the reproductive activity of males and their secretion should have an endocrine control as other sexual secondary organs. J. Morphol. 278:369–379, 2017. © 2017 Wiley Periodicals, Inc.     

Resistance of W/Fu rats to adrenal regeneration hypertension.

The procedure for producing adrenal regeneration hypertension did not cause an increase in the systolic blood pressure of W/Fu animals. The regenerating adrenal gland in W/Fu animals was not restored to normal; reduced numbers of mitochondrial cristae were seen and the mitochondria were smaller in size; regeneration was complete in Sprague-Dawley rats of the Holtzman strain and there was a severe form of hypertensive, cardiovascular disease.     

The comparative morphology of epidermal glands in Pentatomoidea (Heteroptera)

The Heteroptera show a diversity of glands associated with the epidermis. They have multiple roles including the production of noxious scents. Here, we examine the cellular arrangement and cytoskeletal components of the scent glands of pentatomoid Heteroptera in three families, Pentatomidae (stink bugs), Tessaratomidae, and Scutelleridae (shield-backed bugs or jewel bugs). The glands are; (1) the dorsal abdominal glands, (2) the tubular glands of the composite metathoracic gland, and (3) the accessory gland component of the composite metathoracic gland. The dorsal abdominal glands are at their largest in nymphs and decrease in size in adults. The metathoracic gland is an adult-specific gland unit with a reservoir and multiple types of gland cells. The accessory gland is composed of many unicellular glands concentrated in a sinuous line across the reservoir wall. The lateral tubular gland is composed of two-cell units. The dorsal abdominal glands of nymphs are composed of three-cell units with a prominent cuticular component derived from the saccule cell sitting between the duct and receiving canal. The cuticular components that channel secretion from the microvilli of the secretory cell to the exterior differ in the three gland types. The significance of the numbers of cells comprising gland units is related to the role of cells in regenerating the cuticular components of the glands at moulting in nymphs.     

Double NF1 Inactivation Affects Adrenocortical Function in NF1Prx1 Mice and a Human Patient

Background

Neurofibromatosis type I (NF1, MIM#162200) is a relatively frequent genetic condition, which predisposes to tumor formation. Apart from tumors, individuals with NF1 often exhibit endocrine abnormalities such as precocious puberty (2,5–5% of NF1 patients) and some cases of hypertension (16% of NF1 patients). Several cases of adrenal cortex adenomas have been described in NF1 individuals supporting the notion that neurofibromin might play a role in adrenal cortex homeostasis. However, no experimental data were available to prove this hypothesis.

Materials and Methods

We analysed Nf1Prx1 mice and one case of adrenal cortical hyperplasia in a NF1patient.

Results

In Nf1Prx1 mice Nf1 is inactivated in the developing limbs, head mesenchyme as well as in the adrenal gland cortex, but not the adrenal medulla or brain. We show that adrenal gland size is increased in NF1Prx1 mice. Nf1Prx1 female mice showed corticosterone and aldosterone overproduction. Molecular analysis of Nf1 deficient adrenals revealed deregulation of multiple proteins, including steroidogenic acute regulatory protein (StAR), a vital mitochondrial factor promoting transfer of cholesterol into steroid making mitochondria. This was associated with a marked upregulation of MAPK pathway and a female specific increase of cAMP concentration in murine adrenal lysates. Complementarily, we characterized a patient with neurofibromatosis type I with macronodular adrenal hyperplasia with ACTH-independent cortisol overproduction. Comparison of normal control tissue- and adrenal hyperplasia- derived genomic DNA revealed loss of heterozygosity (LOH) of the wild type NF1 allele, showing that biallelic NF1 gene inactivation occurred in the hyperplastic adrenal gland.

Conclusions

Our data suggest that biallelic loss of Nf1 induces autonomous adrenal hyper-activity. We conclude that Nf1 is involved in the regulation of adrenal cortex function in mice and humans.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

The effects of new steroidal anti-androgen, TZP-4238, and chlormadinone acetate on the pituitary, prostate and adrenal gland of the rat: histopathological and immunocytochemical studies.

The atrophic effects of a synthetic steroidal anti-androgen, TZP-4238, on the pituitary, prostate and adrenal gland of rats were investigated. Male Sprague-Dawley rats were divided into three experimental groups. Group 1 consisted of controls. Groups 2 and 3 received chlormadinone acetate (CMA) 50 mg/kg/day and TZP-4238 10 mg/kg/day p.o., respectively, for 3 weeks. CMA (Group 2) produced marked atrophy of the prostate. Furthermore, CMA caused marked atrophy of the adrenal gland. Histopathologically, the remarkable atrophy was observed in the adrenal cortical cells of zonae fasciculata and reticularis. The most striking ultrastructural alterations were noted in the mitochondria. In addition, intramitochondrial localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was less than that in the controls. In the anterior pituitary gland, CMA induced a reduction in the size of ACTH cells. TZP-4238 (Group 3) produced marked atrophy of the prostate. However, TZP-4238 exerted no effect on the adrenal gland or anterior pituitary ACTH cells. In addition, the present histopathological study showed that TZP-4238 or CMA exerted no effect on the testes or anterior pituitary LH cells. Therefore, it is suggested that TZP-4238 causes atrophy of the prostate without any significant histopathological changes in the adrenal glands or anterior pituitary ACTH cells under the present experimental conditions. We further speculated that TZP-4238 had a more potent anti-prostatic effect than CMA and TZP-4238 had a less inhibitory influence than CMA on the pituitary-adrenal axis.     

Effects of nalidixic acid, chloramphenicol, cycloheximide, and anisomycin on structure and development of plastids and mitochondria in greening Euglena gracilis

The substructure of plastids and mitochondria and the alterations caused by the addition of antibiotics were investigated during light-induced proplastid-to-chloroplast transformation in Euglena gracilis. The organisms were grown in presence of the inhibitors up to 3 days (5 generations). Both 40 μg/ml nalidixic acid and 1–1.5 mg/ml chloramphenicol prevent the formation of chloroplasts of normal size and structure by blocking development during early stages. Under our conditions 2 to 5 straight thylakoids are formed beside 1 to 2 girdle-like thylakoids. The former rarely fuse into bands. Non-crystalline prolamellar bodies of considerable size are formed at the distended ends of the plastids in the presence of both drugs. Chloramphenicol also influences mitochondrial size, shape and internal structure. Giant mitochondria can be observed. Nalidixic acid does not change the size and shape of mitochondria, but the matrix frequently appears highly osmiophilic. Cycloheximide in sublethal doses (2–5 μg/ml) or 50 μg/ml anisomycin inhibits plastid development only in the early period after addition. In later culture periods chloroplasts are found enlarged in size with an increased number of thylakoids and bands per organelle. Insertions of new bands are noted at the inner membrane of the chloroplast envelope. The electronmicroscopic observations agree with the results of chloroplast-specific biochemical activities such as light-induced increase in chlorophyll synthesis and of two chloroplast-bound enzyme activities. The results are discussed with respect to metabolic and biogenetic correlations between the two types of organelles in E. gracilis cells.     

GROWTH AND DIFFERENTIATION OF MITOCHONDRIA IN THE REGENERATING RAT ADRENAL CORTEX : A Correlated Biochemical and Stereological Approach

Diameters of the circular profiles of spherical mitochondria in parenchymal cells of the zona fasciculata in rat adrenal cortex were measured for intact controls and for the regenerating adrenal cortex on electron micrographs recorded at random. The diameter data were then processed by Bach's method which deals with the sphere size distribution. The structural parameters of the mitochondria were computed with the aid of an electronic computer. The total number of mitochondria in all the parenchymal cells of the zona fasciculata were calculated. The surface area of the inner mitochondrial membrane was then determined stereologically. Biochemical parameters were obtained for the protein, the phospholipid, and the cytochrome P-450 content, per averaged mitochondrion. The number of cytochrome P-450 molecules contained in the inner membrane was determined in terms of the unit surface area and of the unit amount of phospholipid. These correlated biochemical and stereological parameters have led to the following conclusions. (a) The genesis of the mitochondria after the adrenal enucleation is almost completed within 10 days. (b) During the period of mitochondrial proliferation, the mitochondria are small in size and also immature both in the structure and in the function of their inner membrane, (c) These small and immature mitochondria grow through an increase of the phospholipid and protein, and this increase is accompanied by expansion of the area of the membrane surface, (d) An enrichment of the inner membrane with cytochrome P-450 molecules occurs, thus indicating the differentiation of adrenocortical mitochondria. The process of membrane differentiation is not tightly coupled with that of membrane growth.